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Oxidation of thioanisole and p-methoxythioanisole by lignin peroxidase: kinetic evidence of a direct reaction between compound II and a radical cation.

ABSTRACT: The reaction of H2O2 with thioanisole and p-methoxythioanisole catalysed by lignin peroxidase from Phanerochaete chrysosporium has been studied spectrophotometrically under turnover and single turnover conditions with a stopped-flow apparatus. Pre-formed lignin peroxidase compounds I and II are each able to react with the sulphides to form a sulphide radical cation. The radical cation is then converted into the sulphoxide either by reaction with the medium or by reaction with compound II. This is the first report of a direct reaction between compound II and the substrate radical cation. With thioanisole, significant enantiomeric selectivity and high oxygen incorporation in the sulphoxide are obtained because compound II is preferentially reduced by the enzyme-bound thioanisole radical cation compared with the neutral substrate. By contrast, with p-methoxythioanisole, the data imply formation of an intermediate ternary complex comprising compound II, radical cation and neutral substrate, such that a chain of electron transfer reactions starting from neutral molecule and progressing to oxidized haem via substrate radical cation is facilitated, yielding the native enzyme and two molecules of p-methoxythioanisole radical cation as products. The reactions of compounds I and II with sulphides imply flexing of the apoprotein moiety during catalysis.

SUBMITTER: Bruck TB

PROVIDER: S-EPMC1223635 | biostudies-other | 2003 Sep

REPOSITORIES: biostudies-other

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Project description:BackgroundDespite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility.ResultsTwo water-soluble sulfonated lignins (from Picea abies and Eucalyptus grandis) were chemically characterized and used to estimate single electron-transfer rates to the H2O2-activated Pleurotus eryngii VP (native enzyme and mutated variant) transient states (compounds I and II bearing two- and one-electron deficiencies, respectively). When the rate-limiting reduction of compound II was quantified by stopped-flow rapid spectrophotometry, from fourfold (softwood lignin) to over 100-fold (hardwood lignin) lower electron-transfer efficiencies (k3app values) were observed for the W164S variant at surface Trp164, compared with the native VP. These lignosulfonates have ~20-30 % phenolic units, which could be responsible for the observed residual activity. Therefore, methylated (and acetylated) samples were used in new stopped-flow experiments, where negligible electron transfer to the W164S compound II was found. This revealed that the residual reduction of W164S compound II by native lignin was due to its phenolic moiety. Since both native lignins have a relatively similar phenolic moiety, the higher W164S activity on the softwood lignin could be due to easier access of its mono-methoxylated units for direct oxidation at the heme channel in the absence of the catalytic tryptophan. Moreover, the lower electron transfer rates from the derivatized lignosulfonates to native VP suggest that peroxidase attack starts at the phenolic lignin moiety. In agreement with the transient-state kinetic data, very low structural modification of lignin, as revealed by size-exclusion chromatography and two-dimensional nuclear magnetic resonance, was obtained during steady-state treatment (up to 24 h) of native lignosulfonates with the W164S variant compared with native VP and, more importantly, this activity disappeared when nonphenolic lignosulfonates were used.ConclusionsWe demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs (Trp164 of P. eryngii VPL) is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer.

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Oxidation of thioanisole and p-methoxythioanisole by lignin peroxidase: kinetic evidence of a direct reaction between compound II and a radical cation.

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